Selective serotonin 2a/2c receptor inverse agonists as therapeutics for neurodegenerative diseases

ABSTRACT

Behavioral pharmacological data with the compound of formula (I), a novel and selective 5HT2A/2C receptor inverse agonist, demonstrate in vivo efficacy in models of psychosis and dyskinesias. This includes activity in reversing MK-801 induced locomotor behaviors, suggesting that this compound may be an efficacious anti-psychotic, and activity in an MPTP primate model of dyskinesias, suggesting efficacy as an anti-dyskinesia agent. These data support the hypothesis that 5HT2A/2C receptor inverse agonism may confer antipsychotic and anti-dyskinetic efficacy in humans, and indicate a use of the compound of formula (I) and related agents as novel therapeutics for Parkinson&#39;s Disease, related human neurodegenerative diseases, and psychosis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/759,561, filed Jan. 15, 2005, which claims priority to U.S.Provisional Application No. 60/441,406, filed Jan. 16, 2003, and U.S.Provisional Application No. 60/479,346, filed Jun. 17, 2003, all ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the therapeutic use ofN-(1-methylpiperidin-4-yl)-N-(4-fluorophenylmethyl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamideand related serotonin 2λ/2C receptor inverse agonists to treat a varietyof human neurodegenerative diseases including Parkinson's Disease,Huntington's Disease, Lewy Body Dementia, and Alzheimer's Disease.Specifically, these agents improve motor function in Parkinson'sDisease, and Huntington's Disease. Specifically,N-(1-methylpiperidin-4-yl)-N-(4-fluorophenylmethyl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamideand related compounds can be used to control the behavioral andneuropsychiatric manifestations present in all of these disease states.Pharmaceutical compositions comprised of a combination ofN-(1-methylpiperidin-4-yl)-N-(4-fluorophenylmethyl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamideand existing therapeutic agents are also disclosed.

BACKGROUND OF THE INVENTION

Neurodegenerative disorders (NDs) are a group of related human maladiesthat share a common pathophysiological feature, the progressivedegeneration of selective neuronal populations over the course of time.These neurodegenerative diseases include but are not limited toAlzheimer's Disease and related dementias, Parkinson's Disease,Huntington's Disease, Lewy Body Disease and related movement disorders,and Friedrich's Ataxia and related Spinocerebellar Ataxia's. Each ofthese disorders has unique clinical aspects including age of onset, timecourse of progression, neurological signs and symptoms, neuropsychiatricsymptoms, and sensitivity to known therapeutic agents. In addition, thepathophysiological basis of each of these disorders is caused by geneticmechanisms unique to each disease.

Despite significant progress in elucidating the genetic causesunderlying these disparate disorders, relatively little is known aboutthe biochemical mechanisms that cause the selective neuronaldegeneration common to all of them. In addition, for the most common ofthese disorders, including Parkinson's Disease and Alzheimer's Disease,the genetic factors that cause the rare familial forms of these diseaseshave been discovered, but the pathophysiological basis of the vastmajority of sporadic cases is still unknown. Because of this, nospecific therapeutic agents currently exist that can directly modifydisease progression. Instead, clinicians utilize a variety of existingagents to provide symptomatic relief of the motor, cognitive, andneuropsychiatric manifestations that characterize these disorders. Noneof these existing agents were designed and developed to specificallytreat patients with NDs.

Of the various neurological symptoms that characterize the NDs,abnormalities of motor function, including bradykinesias, dyskinesiasand chorea, and the emergence of neuropsychiatric symptoms, includingpsychosis, and affective symptoms such as anxiety and depression, arecommon and severely impact upon the patient's functional status andquality of life. Unfortunately, most existing therapeutic agents,including antipsychotics and antidepressants, often demonstrateefficacy, yet are very poorly tolerated in these patients. In addition,the available therapeutic agents for Parkinson's Disease, includingL-dopa and dopamine agonists, while generally effective, cause theemergence of severe treatment-limiting side effects that are currentlyintractable to pharmacotherapy.

Multiple factors, both disease and drug related, are primarilyresponsible for the limited tolerability of these agents. First,patients with neurodegenerative disease are particularly sensitive tomost therapeutic agents that are designed to cross the blood-brainbarrier and interact with neuronal targets that confer efficacy againstadverse motoric or neuropsychiatric symptoms. For instance, atypicalantipsychotics are generally well tolerated by healthy volunteers, or inpatients with primary psychiatric disorders like schizophrenia; brainstates that are not characterized by neuronal degeneration. In contrast,when these agents are administered to patients with Parkinson's orHuntington's Disease, they display severe, treatment-limiting adverseeffects on motor function, cause severe sedation, and can worsencognitive functioning. The direct effects of the neuronal losscharacteristic of NDs, and the adaptive changes that occur secondarilyto this are both posited to create a neurochemical and/orneurophysiological state in ND patients that confer this extrasensitivity.

Second, the known mechanisms of action of these drugs, includingantagonism of dopamine receptors, is not tolerated in some patientpopulations secondary to specific alterations in distinct neuronalsystems. For instance, Parkinson's patients have a relatively selectivedegeneration of the ascending dopaminergic neuronal systems, and as aconsequence of this they are deficient in central dopamineneurotransmission. It is therefore not surprising that drugs thatfurther attenuate dopaminergic neurotransmission, by blocking dopaminereceptors, are not well tolerated.

Lastly, nearly all presently known therapeutic agents lack specificityin their mechanisms of action. Antipsychotic and antidepressant drugspossess a multitude of pharmacologically relevant interactions withcritical neuronal proteins including a host of cell surface receptors,ion channels, and re-uptake transporters. This lack of drug targetspecificity is known to confer a variety of adverse effects in non-NDpatient populations, which are qualitatively and quantitatively worse inNI) patients.

These observations highlight the need to develop novel therapeuticagents that are specifically designed to not only demonstrate efficacyagainst these particular disabling symptoms but to also possesstolerability in these specific patient populations. This can be achievedby improving the selectivity of the drug target interactions of newtherapeutic agents. Specifically, the development of agents with novelmechanisms of action that avoid the known pitfalls associated withexisting agents is desired. In addition, improved selectivity can avoidthe known adverse effects associated with interactions with non-efficacyconferring drug targets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plots of D₂ and 5-HT2A receptor agonist activity ofParkinson's Disease therapeutics as determined by the physiologicalypredictive, cell-based, in vivo R-SAT assay. FIG. 1A plots drug activityat human D₂ receptors. FIG. 1B plots drug activity at human Serotonin 2Areceptors.

FIG. 2A is a plot of the efficacy of the compound of formula (I) inreducing MK-801 induced locomotor behaviors in rats against a controlafter s.c. administration over a ten (10) minute time period. FIG. 2B isa plot of the efficacy of the compound of formula (I) in reducing MK-801induced locomotor behaviors in rats against a control after oraladministration over a thirty (30) minute time period.

FIG. 3 shows a bar graph that indicates three dosage levels of thecompound of formula (I) and the effect of each dosage on reducingdyskinesia in a primate model.

FIG. 4 shows the affect of the compound of formula (I) on amphetamineinduced hyperactivity in mice when used in combination with varyingdoses of Haloperidol,

SUMMARY OF THE INVENTION

Disclosed herein is a composition comprising a compound of Formula (I):

and a pharmaceutically acceptable carrier. In some embodiments, thecomposition further comprises an additional therapeutic agent: In someembodiments the additional therapeutic agent is selected from levodopa(SINEMET™, SINEMET-CR™, bromocriptine (PARLODEL™), pergolide (PERMAX™),ephenedrine sulfate (EPHEDRINE™), pemoline CYLERT™), mazindol(SANOREX™), d,l-α-methylphenethylamine (ADDERALL™), methylphenydate(RITALIN™), pramipcxole (MIRAPEX™), modafinil (PROVIGIL™), andropinirole (REQUIP™). In other embodiments, the additional therapeuticagent is an anti-dyskensia agent selected from baclofen (Lioresal™),botulinum toxin (Botox™), clonazepam (Klonopin™), and diazepam(Valium™). In other embodiments, the additional therapeutic agent is ananti-dystonia, anti-myoclonus, or anti-tremor agent selected frombaclofen (LIORESAL™), botulinum toxin (BOTOX™), clonazepam (KLONOPIN™),and diazepam (VALIUM™). In other embodiments, the additional therapeuticagent is an anti-psychotic agent with dopaminergic receptor antagonism.In other embodiments, the additional therapeutic agent is ananti-psychotic agent selected from chlorpromazine (THORAZINE™),haloperodol (HALDOL™), molindone (MOBAN™), thioridazine (MELLARIL™), aphenothiazine, a butyrophenome, diphenulbutylpiperinde (pimozide),thioxanthines (fluphenthixol), substituted benzamides (sulpiride),sertindole, amisulpride, risperidone, clozapine, olanzapine,ziprasidone, aripiprazole, and their active metabolites(N-desmethylclozapine, N-desmethylolanzapine, 9-OH-risperdone)).

Also disclosed herein is a method for treating a neurodegernativedisease comprising: identifying a patient suffering from aneurodegenerative disease and administering to the patient an effectiveamount of an inverse agonist selective for a serotonin receptor; wherebythe dopaminergic therapy associated dyskinesia is reduced. In someembodiments, the neurodegenerative disease is Parkinson's disease,Huntington's disease, Alzheimer's disease, Spinocerebellar Atrophy,Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, LewyBody Dementia, Dystonia, Progressive Supranuclear Palsy, orFrontotemporal Dementia. In one embodiment, the serotonin receptor is a5HT2A receptor. In another embodiment, the serotonin receptor is a 5HT2Creceptor. In some embodiments, the inverse agonist binds to a 5HT2Areceptor or a 5HT2C receptor. In some embodiments, the inverse agonistis the compound of formula (I). One embodiment further comprisesadministering a dopaminergic agent in combination with the compound offormula (I). In some embodiments, the reagent increases dopaminergicactivity and is selected from the group consisting of levodopa,SINAMET™, SINAMETCR™, bromocriptine (PARLODEL™), pergolide (PERMAX™),ephenedrine sulfate (EPHEDRINE™), pemoline CYLERT™), mazindol(SANOREX™), d,l-α-methylphenethylamine (ADDERALL™), methylphenydate(RITALIN™), pramipexole (MIRAPEX™), modafinil (PROVIGIL™) and ropinirole(REQUIP™).

Also disclosed herein is, a method for treating dyskinesia associatedwith dopaminergic therapy comprising: identifying a patient sufferingfrom dopaminergic therapy associated dyskinesia and administering to thepatient an effective amount of an inverse agonist selective for aserotonin receptor; whereby the dopaminergic therapy associateddyskinesia is reduced. In one embodiment the serotonin receptor is a5HT2A receptor. In another embodiment the serotonin receptor is a 5HT2Creceptor. In some embodiments, the inverse agonist binds to a 5HT2Areceptor and a 5HT2C receptor. In one embodiment, the inverse agonist isthe compound of formula (I). Some embodiments further compriseadministering an anti-dyskensia agent in combination with the compoundof formula (I). In some embodiments, the anti-dyskinesia agent isselected from the group consisting of baclofen (Lioresal™), botulinumtoxin (Botox™), clonazepam (Klonopin™), and diazepam (Valium™). In someembodiments, the patient suffers from a neurodegenerative diseaseselected from the group consisting of Parkinson's disease, Huntington'sdisease, Alzheimer's disease, Spinocerebellar Atrophy, Tourette'sSyndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy BodyDementia, Dystonia, Progressive Supranuclear Palsy, and FrontotemporalDementia.

Further disclosed herein is a method for treating dystonia, myoclonus,or tremor associated with dopaminergic therapy comprising: identifying apatient suffering from dopaminergic therapy associated dystonia,myoclonus, or tremor; and administering to the patient an effectiveamount of an inverse agonist selective for a serotonin receptor; wherebythe dopaminergic therapy associated dystonia, myoclonus, or tremor isreduced. In one embodiment the serotonin receptor is a 5HT2A receptor.In another embodiment, the serotonin receptor is a 5HT2C receptor. Insome embodiments, the inverse agonist binds to a 5HT2A receptor and a5HT2C receptor. In some embodiments, the inverse agonist is the compoundof formula (I). Some embodiments further comprise an anti-dystonia,anti-myoclonus, or anti-tremor agent in combination with the compound offormula (I). In some embodiments, the anti-dystonia, anti-myoclonus, oranti-tremor agent is selected from the group consisting of baclofen(LIORESAL™), botulinum toxin (BOTOX™), clonazepam (KLONOPIN™), anddiazepam (VALIUM™).

Also disclosed herein is a method for treating psychosis associated withdopaminergic therapy comprising: identifying a patient suffering fromdopaminergic therapy associated psychosis; and administering to thepatient an effective amount of an inverse agonist selective for aserotonin receptor; whereby symptoms of dopaminergic therapy associatedpsychosis is reduced. In one embodiment the serotonin receptor is a 5H2Areceptor. In another embodiment the serotonin receptor is a 5HT2Creceptor. In some embodiments the inverse agonist binds to a 5HT2Areceptor and a 5HT2C receptor. In some embodiments the inverse agonistis the compound of formula (I). Some embodiments further comprise ananti-psychotic agent in combination with the compound of formula (I). Insome embodiments, the anti-psychotic agent is selected from the groupconsisting of chlorpromazine (THORAZINE™), haloperodol (HALDOL™),molindone (MOBAN™), thioridazine (MELLARIL™), a phenothiazine, abutyrophenome, diphenulbutylpiperinde (pimozide), thioxanthines(fluphenthixol), substituted benzamides (sulpiride), sertindole,amisulpride, risperidone, clozapine, olanzapine, ziprasidone,aripiprazole, and their active metabolites (N-desmethylclozapine,N-desmethylolanzapine, 9-OH-risperdone)). In some embodiments, thepatient suffers from a neurodegenerative disease selected from the groupconsisting of Parkinson's disease, Huntington's disease, Alzheimer'sdisease, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich'sAtaxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia,Progressive Supranuclear Palsy, and Frontotemporal Dementia.

Also disclosed herein is a method for treating a neuropsyhiatric diseasecomprising: identifying a patient suffering from a neuropsyhiatricdisease; and administering to the patient an effective amount of aninverse agonist selective for a serotonin receptor. In some embodiments,the neuropsychiatric disease is selected from the group consisting ofschizophrenia, schizoaffective disorders, mania, behavioral disturbancesassociated with dementia and psychotic depression. In some embodimentsthe serotonin receptor is a 5HT2A receptor. In some embodiments theserotonin receptor is a 5HT2C receptor. In some embodiments the inverseagonist binds to a 5HT2A receptor or a 5HT2C receptor. In oneembodiment, the inverse agonist is the compound of formula (I). Someembodiments further comprise administering an antipsychotic agent incombination with the inverse agonist, the anti-psychotic agent selectedfrom the group consisting of chlorpromazine (THORAZINE™), haloperodol(HALDOL™), molindone (MOBAN™), thioridazine (MELLARIL™), aphenothiazine, a butyrophenome, diphenulbutylpiperinde (pimozide),thioxanthines (fluphenthixol), substituted benzamides (sulpiride),sertindole, amisulpride, risperidone, clozapine, olanzapine,ziprasidone, aripiprazole, and their active metabolites(N-desmethylclozapine, N-desmethylolanzapine, 9-OH-risperdone)).

Also disclosed herein is a compound having the structure of Formula (I):

Additionally disclosed herein is a method of inhibiting an activity of amonoamine receptor comprising contacting the monoamine receptor or asystem containing the monoamine receptor with an amount of the compoundof formula (I) that is effective in inhibiting the activity of themonoamine receptor. In some embodiments, the monoamine receptor is aserotonin receptor. In one embodiment the serotonin receptor is the5-HT2A subclass. In some embodiments the serotonin receptor is in thecentral nervous system. In some embodiments the serotonin receptor is inthe peripheral nervous system. In some embodiments the serotoninreceptor is in blood cells or platelets. In some embodiments theserotonin receptor is mutated or modified. In some embodiments theactivity is signaling activity. In some embodiments the activity isconstitutive. In some embodiments the activity is associated withserotonin receptor activation.

Also disclosed herein is a method of inhibiting an activation of amonoamine receptor comprising contacting the monoamine receptor or asystem containing the monoamine receptor with an amount of the compoundof formula (I) that is effective in inhibiting the activation of themonoamine receptor. In some embodiments, the activation is by anagonistic agent. In some embodiments the agonistic agent is exogenous.In some embodiments the agonistic agent is endogenous. In someembodiments the activation is constitutive. In some embodiments themonoamine receptor is a serotonin receptor. In some embodiments theserotonin receptor is the 5HT2A subclass. In some embodiments theserotonin receptor is in the central nervous system. In some embodimentsthe serotonin receptor is in the peripheral nervous system. In someembodiments the serotonin receptor is in blood cells or platelets. Insome embodiments the serotonin receptor is mutated or modified.

Also disclosed herein is a method of treating a disease conditionassociated with a monoamine receptor comprising administering to asubject in need of such treatment a therapeutically effective amount ofthe compound of formula (I). In some embodiments the disease conditionis selected from the group consisting of schizophrenia, psychosis,migraine, hypertension, thrombosis, vasospasm, ischemia, depression,anxiety, sleep disorders and appetite disorders. In some embodiments thedisease condition is associated with dysfunction of a monoaminereceptor. In some embodiments, the disease condition is associated withactivation of a monoamine receptor. In some embodiments, the diseasecondition is associated with increased activity of monoamine receptor.In some embodiments, the monoamine receptor is a serotonin receptor. Insome embodiments the serotonin receptor is the 5-HT2A subclass. In someembodiments the serotonin receptor is in the central nervous system. Insome embodiments the serotonin receptor is in the peripheral nervoussystem. In some embodiments the serotonin receptor is in blood cells orplatelets. In some embodiments, the serotonin receptor is mutated ormodified.

Also disclosed herein is a method of treating schizophrenia comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount the compound of formula (I).

Also disclosed herein is a method of treating migraine comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of the compound of formula (I).

Also disclosed herein is a method of treating psychosis comprisingadministering to a subject in need of such treatment a therapeuticallyeffective amount of the compound of formula (I).

Also disclosed herein is a method for identifying a genetic polymorphismpredisposing a subject to being responsive the compound of formula (I),comprising: administering to a subject a therapeutically effectiveamount of said compound; measuring the response of said subject to saidcompound, thereby identifying a responsive subject having an ameliorateddisease condition associated with a monoamine receptor; and identifyinga genetic polymorphism in the responsive subject, wherein the geneticpolymorphism predisposes a subject to being responsive to said compound.In some embodiments the ameliorated disease condition is associated withthe 5-HT class or 5-HT2A subclass of monoaminergic receptors.

Additionally disclosed herein is a method for identifying a subjectsuitable for treatment with the compound of Formula (I), comprisingdetecting the presence of a polymorphism in a subject wherein thepolymorphism predisposes the subject to being responsive to thecompound, and wherein the presence of the polymorphism indicates thatthe subject is suitable for treatment with the compound of formula (I).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

For the purpose of the current disclosure, the following definitionsshall in their entireties be used to define technical terms, and shallalso, in their entireties, be used to define the scope of thecomposition of matter for which protection is sought in the claims.

“Constitutive activity” is defined as the elevated basal activity of areceptor that is independent of the presence of an agonist. Constitutiveactivity of a receptor may be measured using a number of differentmethods, including cellular (e.g., membrane) preparations (see, e.g.,Barr &. Manning, J. Biol. Chem. 272:32979-87 (1997)), purifiedreconstituted receptors with, or without the associated G-protein inphospholipid vesicles (Cerione et al., Biochemistry 23:4519-25 (1984)),and functional cellular assays (U.S. Patent Application Ser. No.60/103,317) or any other method known in the art.

“Agonist” is defined as a compound that increases the basal activity ofa receptor when it contacts the receptor.

An “antagonist” is defined as a compound that competes with an agonistor inverse agonist for binding to a receptor, thereby blocking theaction of an agonist or inverse agonist on the receptor. However, anantagonist (also known as a “neutral” antagonist) has no effect onconstitutive receptor activity.

An “inverse agonist” is defined as a compound that decreases the basalactivity of a receptor (i.e., signaling mediated by the receptor). Suchcompounds are also known as negative antagonists. An inverse agonist isa ligand for a receptor that causes the receptor to adopt an inactivestate relative to a basal state occurring in the absence of any ligand.Thus, while an antagonist can inhibit the activity of an agonist, aninverse agonist is a ligand that can alter the conformation of thereceptor in the absence of an agonist. The concept of an inverse agonisthas been explored by Bond et al. in Nature 374:272 (1995). Morespecifically, Bond et al. have proposed β₂-adrenoceptor exists in anequilibrium between an inactive conformation and a spontaneously activeconformation. Agonists are proposed to stabilize the receptor in anactive conformation. Conversely, inverse agonists are believed tostabilize an inactive receptor conformation. Thus, while an antagonistmanifests its activity by virtue of inhibiting an agonist, an inverseagonist can additionally manifest its activity in the absence of anagonist by inhibiting the spontaneous conversion of an unligandedreceptor to an active conformation.

The “5-HT2A receptor” is defined as a receptor, having an activitycorresponding to the activity of the human serotonin receptor subtype,which was characterized through molecular cloning and pharmacology asdetailed in Saltzman et al., Biochem. Biophys. Res. Comm. 181:1469-78;and Julius et al., Proc. Natl. Acad. Sci. USA 87:928-932, thedisclosures of which are incorporated herein by reference in theirentireties.

The term “subject” refers to an animal, preferably a mammal, mostpreferably a human, who is the object of treatment, observation orexperiment.

“Selective” is defined as a property of a compound whereby an amount ofthe compound sufficient to effect a desired response from a particularreceptor type, subtype, class or subclass with significantly less orsubstantially little or no effect upon the activity other receptortypes. For example, a selective compound may have at least a 10-foldgreater effect on activity of the desired receptor than on otherreceptor types. In some cases, a selective compound may have at least a20-fold greater effect on activity of the desired receptor than on otherreceptor types, or at least a 50-fold greater effect, or at least a100-fold greater effect, or at least a 1000-fold greater effect, or atleast a 10,000-fold greater effect, or at least a 100,000-fold greatereffect, or more than a 100,000-fold greater effect.

“Selectivity” or “selective,” as an inverse agonist is understood as aproperty of the compound of the invention whereby an amount of compoundthat effectively inversely agonizes the 5-HT2A receptor, and therebydecreases its activity, causes little or no inverse agonistic orantagonistic activity at other, related or unrelated, receptors. Inparticular, in one embodiment, a compound has surprisingly been foundnot to interact strongly with other serotonin receptors (5-HT 1A, 1B,1D, 1E, 1F, 2B, 2C, 4A, 6, and 7) at concentrations where the signalingof the 5HT2A receptor is strongly or completely inhibited. In oneembodiment, the compound is also selective with respect to othermonoamine-binding receptors, such as the dopaminergic, histaminergic,adrenergic and muscarinic receptors. Compounds that are highly selectivefor 5-HT2A receptors may have a beneficial effect in the treatment ofpsychosis, schizophrenia or similar neuropsychiatric disorders, whileavoiding adverse effects associated with drugs hitherto suggested forthis purpose.

Some embodiments described herein relate to serotonin 2A or 2C receptorinverse agonists, including compositions and methods for treatingcertain side-effects caused or exacerbated by dopaminergenicagent-associated therapies commonly used in treating neurodegenerativediseases. For example, the compounds disclosed herein have utility inreducing dyskinesia and psychosis associated with dopaminergenictherapies used in treating Parkinson's disease, a neurodegenerativedisease. According to one embodiment, the compoundN-(1-methylpiperidin-4-yl)-N-(4-fluorophenylmethyl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamidehaving the structure of formula (I) is provided:

One embodiment relates to a composition comprising the compound offormula (I) and a pharmaceutically acceptable carrier. The compositionmay also contain other compounds such as compounds for treatingdyskensia, dystonia, or psychosis.

According to one embodiment, the tartrate salt of the compound,N-(1-methylpiperidin-4-yl)-N-(4-fluorophenylmethyl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamideis a potent, selective, orally bioavailable 5-HT2A receptor inverseagonist. The compound of formula (I) also possesses lesser potency as a5-HT2C receptor inverse agonist and lacks intrinsic activity at theremaining monoaminergic receptor subtypes. Perhaps most notably, thecompound of formula (I) lacks activity at dopamine receptor subtypes.(See U.S. patent application Ser. No. 09/800,096, which is herebyincorporated by reference in its entirety). Extensive behavioralpharmacological profiling of the compound of formula (I), includingpre-clinical models of antipsychotic and anti-dyskinetic drug actions,support the therapeutic use of the compound in Parkinson's Disease andrelated human neurodegenerative diseases.

Parkinson's Disease (PD) is a common and progressive neurodegenerativedisease. Current estimates suggest that nearly 900,000 individuals inthe United States have PD and that the prevalence is increasing as theUS population ages. Dopamine receptor agonists are used to alleviate thesymptoms of PD, such as motoric dysfunction. Unfortunately, theprotracted use of these dopaminergenic agents causes, over time,neuropsychiatric (psychosis) and troublesome motor (dyskinesia) sideeffects in 30 to 80% of patients, respectively.

Antipsychotics and dopamine receptor antagonists can be effective inameliorating these adverse effects. Unfortunately, many of thesecompounds significantly worsen motor function in PD patients secondaryto their hypo-dopaminergic state. Biochemical and pharmacological datasupport the hypothesis that potentiation of serotonergicneurotransmission may be pathophysiologically related to the developmentof dyskinesias and psychosis in these patients. While not being bound bythis theory, the compounds disclosed herein were selected to exploit therelationship of serotonergic activity and the negative side-effectsassociated with dopaminergenic therapy.

L-dopa is a typical dopaminergic compound used to treat PD. L-dopa hasbeen shown to increase central serotonin release, turnover, andmetabolite concentrations in rodent brain. Direct acting dopaminereceptor agonists like pergolide possess, in additional to theirdopamine receptor agonist properties, potent agonist activity atserotonin 2A (5-HT2A) and 2C (5-HT2C) receptors as demonstrated byvarious in vitro pharmacological assays.

In one embodiment, the compounds disclosed herein can be used to treatmany side-effects that arise from dopaminergenic therapy. For example,the disclosed compounds are also useful for treatment of dyskinesia orpsychosis caused or exacerbated as a side-effect of other therapeuticagents such as L-dopa. In one embodiment, the compounds are preferablyused for the treatment of dyskinesia or psychosis associated with L-dopatreatment.

The compounds may be used to treat existing dyskinesia or psychosis ormay be used prophylactic fashion when for example, it is considerednecessary to initiate L-dopa therapy and it is feared that dyskinesia orpsychosis may develop.

The compounds may be used to treat dyskinesia or psychosis as amonotherapy or as an adjunct to medicaments to prevent or treatdyskinesia or psychosis side-effects caused by the medicament oralternatively the compounds may be given in combination with othercompounds which also reduce dyskinesia.

In some embodiments, the compounds described herein can be formulatedinto compositions for administration to patients in need thereof.Appropriate compositions can take a number of different forms dependingon how the composition is to be used. For example, the composition maybe in the form of a powder, tablet, capsule, liquid, ointment, cream,gel, hydrogel, aerosol, spray, micelle, liposome or any otherpharmaceutically acceptable form. One of ordinary skill in the art wouldreadily appreciate that an appropriate vehicle for use with thedisclosed compounds of the invention should be one that is welltolerated by a recipient of the composition. The vehicle should alsoreadily enable the delivery of the compounds to appropriate targetreceptors. For example, one of ordinary skill in the art would know toconsult Pharmaceutical Dosage Forms and Drug Delivery Systems, by Ansel,et al., Lippincott Williams & Wilkins Publishers; 7th ed. (1999) or asimilar text for guidance regarding such formulations.

The composition of the invention may be used in a number of ways. Forinstance, systemic administration may be required in which case thedisclosed compounds can be formulated into a composition that can beingested orally in the form of a tablet, capsule or liquid.Alternatively the composition may be administered by injection into theblood stream. Injections may be intravenous (bolus or infusion) orsubcutaneous (bolus or infusion). The disclosed compounds can also beadministered centrally by means of intracerebral,intracerebroventricular, or intrathecal delivery.

The compound may also be used with a time delayed release device. Suchdevices may, for example, be inserted under the skin and the compoundmay be released over weeks or months. Such a device may be particularlyuseful for patients with long term dyskinesia such as patients oncontinuous L-dopa therapy for the treatment of PD. The devices may beparticularly advantageous when a compound is used which would normallyrequire frequent administration (e.g., frequent injection).

It will be readily appreciated that the amount of a compound required isdetermined by biological activity and bioavailability which in turndepends on the mode of administration, the physicochemical properties ofthe compound employed and whether the compound is being used as amonotherapy or in a combined therapy. The frequency of administrationwill also be influenced by the above mentioned factors and particularlythe half-life of the compound within the subject being treated.

One of ordinary skill in the art would appreciate that specificformulations of compositions and precise therapeutic regimes (such asdaily doses of the compounds and the frequency of administration) can bedetermined using known procedures. Such procedures conventionallyemployed by the pharmaceutical industry include in vivo experimentationand clinical trials.

Generally, a daily dose of between 0.01 μg/kg of body weight and 1.0g/kg of body weight of a serotonin 2λ/2C receptor inverse agonist can beused with the methods disclosed herein. In one embodiment, the dailydose is between 0.01 mg/kg of body weight and 100 mg/kg of body weight,or any milligram or half-milligram quantity in this disclosed range,e.g., 1.5, 2, 2.5, etc.

Daily doses may be given as a single administration (e.g. a daily tabletfor oral consumption or as a single daily injection). Alternatively thecompound used may require administration twice or more times during aday, depending on the kinetics of the drug associated with theindividual patient. Alternatively a slow release device may be used toprovide optimal doses to a patient without the need to administerrepeated doses.

Biochemical Evidence

The cornerstone of current pharmacological intervention in PD remainsL-dopa based therapies. L-dopa readily crosses the blood brain barrier,is taken up by neurons and undergoes rapid enzymatic conversion todopamine, via L-aromatic acid decarboxylase (LAAD) activity indopaminergic neurons. The increased availability and release of dopaminefrom these neurons clearly leads to increased dopaminergic transmission,and clinical efficacy in reversing the motoric effects of thehypo-dopaminergic state observed in PD. However, L-dopa lacksspecificity for dopaminergic systems, and LAAD is widely expressed inbrain. Early biochemical observations in rat brain noted that L-dopasubstantially reduced central serotonergic stores, and increased theconcentration of the principle serotonin metabolite of5-hydroxyindoleacetic acid (5-HIAA) (1). Histochemical approaches havedemonstrated that L-dopa accumulates in serotonergic neurons, andneurotransmitter release experiments have demonstrated that L-dopamarkedly increased the release of both dopamine and serotonin, thatrelease of serotonin is dependent upon LAAD activity, and that it is noteliminated by the selective destruction of dopaminergic neurons (2,3).These observations suggest that the administration of L-dopa to PDpatients results in marked increases in the release of centralserotonin, potentiating serotonergic neurotransmission. Finally,post-mortem biochemical analysis of PD patients that developedpsychosis, when compared to a matched group that did not developneuropsychiatric disturbances, found that the patients with psychosishad significant elevations in serotonin and 5-HIAA levels in multiplecortical and sub-cortical structures, most notably various mesencephalicnuclei including the red nucleus (4).

Serotonin or 5-hydroxytryptamine (5-HT) plays a significant role in thefunctioning of the mammalian body. In the central nervous system, 5-HTis an important neurotransmitter and neuromodulator that is implicatedin such diverse behaviors and responses as sleeping, eating, locomotion,perceiving pain, learning and memory, sexual behavior, controlling bodytemperature and blood pressure. In the spinal column, serotonin plays animportant role in the control systems of the afferent peripheralnociceptors (Moulignier, Rev. Neurol. 150:3-15, (1994)). Peripheralfunctions in the cardiovascular, hematological, and gastrointestinalsystems have also been ascribed to 5-HT. 5-HT has been found to mediatea variety of contractile, secretory, and electrophysiologic effectsincluding vascular and nonvascular smooth muscle contraction, andplatelet aggregation. (Fuller, Biology of Serotonergic Transmission,1982; Botillin, Serotonin In Mental Abnormalities 1:316 (1978); Barchas,et al., Serotonin and Behavior, (1973)). The 5-HT2A receptor subtype(also referred to as subclass) is widely yet discretely expressed in thehuman brain, including many cortical, limbic, and forebrain regionspostulated to be involved in the modulation of higher cognitive andaffective functions. This receptor subtype is also expressed on matureplatelets where it mediates, in part, platelet aggregation, one of theinitial steps in the process of vascular thrombosis.

Given the broad distribution of serotonin within the body, it isunderstandable that tremendous interest in drugs that affectserotonergic systems exists (Gershon, et at, The Peripheral Actions of5-Hydroxytryptamine, 246 (1989); Saxena, et at, J. CardiovascularPharmacol. 15: Supp. 7 (1990)). Serotonin receptors are members of alarge human gene family of membrane-spanning proteins that function astransducers of intercellular communication. They exist on the surface ofvarious cell types, including neurons and platelets, where, upon theiractivation by either their endogenous ligand serotonin or exogenouslyadministered drugs, they change their conformational structure andsubsequently interact with downstream mediators of cellular signaling.Many of these receptors, including the 5-HT2A subclass, are O-proteincoupled receptors (GPCRs) that signal by activating guanine nucleotidebinding proteins (G-proteins), resulting in the generation, orinhibition of, second messenger molecules such as cyclic AMP, inositolphosphates, and diacylglycerol. These second messengers then modulatethe function of a variety of intracellular enzymes, including kinasesand ion channels, which ultimately affect cellular excitability andfunction.

At least 15 genetically distinct 5-HT receptor subtypes have beenidentified and assigned to one of seven families (5-HT1-7). Each subtypedisplays a unique distribution, preference for various ligands, andfunctional correlate(s). Serotonin may be an important component invarious types of pathological conditions such as certain psychiatricdisorders (depression, aggressiveness, panic attacks, obsessivecompulsive disorders, psychosis, schizophrenia, suicidal tendency),certain neurodegenerative disorders (Alzheimer-type dementia,Parkinsonism, Huntington's chorea), anorexia, bulimia, disordersassociated with alcoholism, cerebral vascular accidents, and migraine(Meltzer, Neuropsychopharmacology, 21:106 S-115S (1999); Barnes & Sharp,Neuropharmacology, 38:1083-1152 (1999); Glennon, Neurosci. BiobehavioralRev., 14:35 (1990)). Recent evidence strongly implicates the 5-HT2receptor subtype in the etiology of such medical conditions ashypertension, thrombosis, migraine, vasospasm, ischemia, depression,anxiety, psychosis, schizophrenia, sleep disorders and appetitedisorders.

Schizophrenia is a particularly devastating neuropsychiatric disorderthat affects approximately 1% of the human population. It has beenestimated that the total financial cost for the diagnosis, treatment,and lost societal productivity of individuals affected by this diseaseexceeds 2% of the gross national product (GNP) of the United States.Current treatment primarily involves pharmacotherapy with a class ofdrugs known, as antipsychotics. Antipsychotics are effective inameliorating positive symptoms (e.g., hallucinations and delusions), yetthey frequently do not improve negative symptoms (e.g., social andemotional withdrawal, apathy, and poverty of speech).

Currently, nine major classes of antipsychotics are prescribed to treatpsychotic symptoms. Use of these compounds is limited, however, by theirside effect profiles. Nearly all of the “typical” or older generationcompounds have significant adverse effects on human motor function.These “extrapyramidal” side effects, so termed due to their effects onmodulatory human motor systems, can be both acute (e.g., dystonicreactions, a potentially life threatening but rare neuroleptic malignantsyndrome) and chronic (e.g., akathisias, tremors, and tardivedyskinesia). Drug development efforts have, therefore, focused on newer“atypical” agents free of these adverse effects.

Antipsychotic drugs have been shown to interact with a large number ofcentral monoaminergic neurotransmitter receptors, includingdopaminergic, serotonergic, adrenergic, muscarinic, and histaminergicreceptors. It is likely that the therapeutic and adverse effects ofthese drugs are mediated by distinct receptor subtypes. The high degreeof genetic and pharmacological homology between these receptor subtypeshas hampered the development of subtype-selective compounds, as well asthe determination of the normal physiologic or pathophysiologic role ofany particular receptor subtype. Thus there is a need to develop drugsthat are selective for individual receptor classes and subclassesamongst monoaminergic neurotransmitter receptors.

The prevailing theory for the mechanism of action of antipsychotic drugsinvolves antagonism of dopamine D2 receptors. Unfortunately, it islikely that antagonism of dopamine D2 receptors also mediates theextrapyramidal side effects. Antagonism of 5-HT2A is an alternatemolecular mechanism for drugs with antipsychotic efficacy, possiblythrough antagonism of heightened or exaggerated signal transductionthrough serotonergic systems. 5-HT2A antagonists are therefore goodcandidates for treating psychosis without extrapyramidal side effects.

Traditionally, these receptors have been assumed to exist in a quiescentstate unless activated by the binding of an agonist (a drug thatactivates a receptor). It is now appreciated that many, if not most, ofthe GPCR monoamine receptors, including serotonin receptors, can existin a partially activated state in the absence of their endogenousagonists. This increased basal activity (constitutive activity) can beinhibited by compounds called inverse agonists. Both agonists andinverse agonists possess intrinsic activity at a receptor, in that theyalone can activate or inactivate these molecules, respectively. Incontrast, classic or neutral antagonists compete against agonists andinverse agonists for access to the receptor, but do not possess theintrinsic ability to inhibit elevated basal or constitutivereceptor-responses.

We have elucidated an important aspect of 5-HT2A receptor function byapplying the Receptor Selection and Amplification Technology (U.S. Pat.No. 5,707,798, 1998; Chem. Abstr. 128; 111548 (1998) and citationstherein), to the study of the 5-HT2 subclass of serotonin receptors.R-SAT is a phenotypic assay of receptor function that involves theheterologous expression of receptors in mammalian fibroblasts. Usingthis technology we were able to demonstrate that native 5-HT2A receptorspossess significant constitutive, or agonist-independent, receptoractivity (U.S. Patent Application Ser. No. 60/103,317, hereinincorporated by reference). Furthermore, by directly testing a largenumber of centrally acting medicinal compounds with known clinicalactivity in neuropsychiatric disease, we determined that compounds withantipsychotic efficacy all shared a common molecular property. Nearlyall of these compounds, which are used by psychiatrists to treatpsychosis, were found to be potent 5-HT2A inverse agonists. This uniqueclinico-pharmacologic correlation at a single receptor subtype iscompelling evidence that 5-HT2A receptor inverse agonism is a molecularmechanism of antipsychotic efficacy in humans.

Detailed pharmacological characterization of a large number ofantipsychotic compounds revealed that they possess broad activity atmultiple related receptor subtypes. Most of these compounds displayagonist, competitive antagonist, or inverse agonist activity at multiplemonoaminergic receptor subtypes, including serotoninergic, dopaminergic,adrenergic, muscarinic and histaminergic receptors. This broad activityis likely responsible for the sedating, hypotensive, and motor sideeffects of these compounds. It would therefore be of great advantage todevelop compounds that are selective inverse agonists of the 5-HT2Areceptor, but which have little or no activity on other monaminereceptor subtypes, especially dopamine D2 receptors. Such compounds maybe useful in the treatment of human disease (e.g., as anti-psychotics),and may avoid the adverse side effects associated with non-selectivereceptor interactions.

The compound of formula (I) is active at monoamine receptors,specifically serotonin receptors. In one embodiment, the compound actsas inverse agonist at the 5-HT2A receptor. Thus, experiments performedon cells transiently expressing the human phenotype of said receptorhave shown that the compound of formula (I) attenuates the signaling ofsuch receptors in the absence of additional ligands acting upon thereceptor.

The compound has thus been found to possess intrinsic activity at thisreceptor and is able to attenuate the basal, non-agonist-stimulated,constitutive signaling responses that the 5-HT2A receptor displays. Theobservation that the compound of formula (I) is an inverse agonist alsoindicates that the compound has the ability to antagonize the activationof 5-HT2A receptors that is mediated by endogenous agonists or exogenoussynthetic agonist ligands.

In one embodiment, the compound of formula (I) shows a relatively highdegree of selectivity towards the 5-HT2A subtype of serotonin receptorsrelative to other subtypes of the serotonin (5-HT) family of receptorsas well is to other receptors, most particularly the monoaminergicG-protein coupled receptors, such as dopamine receptors.

The compound of formula (I) may therefore be useful for treating oralleviating symptoms of disease conditions associated with impairedfunction, in particular elevated levels of activity, of especially5-HT2A receptors, whether this impaired function is associated withimproper levels of receptor stimulation or phenotypical aberrations.

Others have previously hypothesized that certain neuropyschologicaldiseases might be caused by altered levels of constitutive activity ofmonoamine receptors. Such constitutive activity might be modified viacontacting the relevant receptor with a synthetic inverse agonist. Bydirectly testing a large number of centrally acting medicinal compoundswith known clinical activity in neuropsychiatric disease, we determinedthat compounds with antipsychotic efficacy all shared a common molecularproperty. Nearly all of these compounds that are used by psychiatriststo treat psychosis were found to be potent 5-HT2A inverse agonists. Thiscorrelation is compelling evidence that 5-HT2A receptor inverse agonismis a molecular mechanism of antipsychotic efficacy in humans.

Detailed pharmacological characterization of a large number ofantipsychotic compounds in our laboratory revealed that they possessbroad activity at multiple related receptor subtypes. Most of thesecompounds display either agonist, competitive antagonist, or inverseagonist activity at multiple monoaminergic receptor subtypes includingserotoninergic, dopaminergic, adrenergic, muscarinic and histaminergicreceptors. This broad activity is likely responsible for the sedating,hypotensive, and motor side effects of these compounds. In oneembodiment, the compound of formula (I) possesses efficacy as, forexample, a novel antipsychotic, but will have fewer or less severe sideeffects than existing compounds.

In one embodiment a method is provided to inhibit activity of amonoamine receptor. This method comprises contacting a monoaminereceptor or a system containing the monamine receptor, with an effectiveamount of the compound of formula (I). According to one embodiment, themonamine receptor is a serotonin receptor. In one embodiment, thecompound is selective for the 5-HT2A receptor subclass. In anotherembodiment, the compound has little or substantially no activity toother types of receptors, including other serotonergic receptors andmost particularly, monoaminergic G-protein coupled receptors, such asdopaminergic receptors.

The system containing the monoamine receptor may, for example, be asubject such as a mammal, non-human primate or a human. The receptor maybe located in the central or peripheral nervous system, blood cells orplatelets.

The system may also be an in vivo or in vitro experimental model, suchas a cell culture model system that expresses a monamine receptor, acell-free extract thereof that contains a monoamine receptor, or apurified receptor. Non-limiting examples of such systems are tissueculture cells expressing the receptor or extracts or lysates thereof.Cells that may be used in the present method include any cells capableof mediating signal transduction via monoamine receptors, especially the5HT2A receptor, either via endogenous expression of this receptor (e.g.,certain types of neuronal cells lines, for example, natively express the5-HT2A receptor), or following transfection of cells with plasmidscontaining the receptor gene. Such cells are typically mammalian cells(or other eukaryotic cells, such as insect cells or Xenopus oocytes),because cells of lower organisms generally lack the appropriate signaltransduction pathways for the present purpose. Examples of suitablecells include: the mouse fibroblast cell line NIH 3T3 (ATCC CRL 1658),which responds to transfected 5HT2A receptors by stimulating growth; RAT1 cells (Pace et al., Proc. Natl. Acad. Sci. USA 88:7031-35 (1991)); andpituitary cells (Vallar et al., Nature 330:556-58 (1987). Other usefulmammalian cells for the present method include HEK 293 cells, CHO cells,and COS cells.

One embodiment provides methods of inhibiting activity of a native,mutated or modified monoamine receptor. Also provided are kits forperforming the same. In one embodiment, the activity of the receptor isa signaling activity. In another embodiment, the activity of thereceptor is the constitutive basal activity of the receptor.

In one embodiment, the activity of the receptor is a response, such as asignaling response, to an endogenous agonist, such as 5-HT, or anexogenous agonistic agent, such as a drug or other synthetic ligand. Thecompound of formula (I) may act by either inversely agonizing orantagonizing the receptor.

In one embodiment, the compound of formula (I) is an inverse agonistselective for the 5-HT2A receptor and the compound has little orsubstantially no activity toward other serotonergic or othermonoaminergic receptors, such as dopaminergic receptors.

In a further embodiment, a method is provided for inhibiting anactivation of a monoamine receptor comprising contacting the monoaminereceptor, or a system containing the monoamine receptor, with thecompound of formula (I). The activation of the receptor may be due to anexogenous or endogenous agonist agent, or may be the constitutiveactivation associated with a native, mutated or modified receptor. Thereceptor may be purified or present in an in vitro or in vivo system.The receptor may also be present in the central or peripheral nervoussystem, blood cells or platelets of a nonhuman or human subject. Alsoprovided are kits for performing the same.

In one embodiment, the compound of formula (I) is selective for 5-HTclass serotonin receptors, such as the 5-HT2A subclass of serotoninreceptors. In another embodiment, the compound has little orsubstantially no anti-dopaminergic activity.

One embodiment provides methods of treating a disease conditionassociated with a monoamine receptor comprising administering to amammal in need of such treatment an effective amount of the compound offormula (I). One embodiment provides methods for treating or alleviatingdisease conditions associated with improper function or stimulation ofnative, as well as mutated or otherwise modified, forms of centralserotonin receptors, particularly the 5-HT class of such receptors,comprising administration of an effective amount of a selective inverseagonist of formula (I) to a host in need of such treatment. Alsoprovided are kits for performing the same.

In one embodiment, the receptor is the 5-HT2A subclass. In oneembodiment, the disease condition is associated with dysfunction of theserotonin receptor.

In another embodiment, the disease condition is associated withactivation of the serotonin receptor, for instance, inappropriatelyelevated or constitutive activation, elevated serotonergic tone, as wellas disease conditions associated with secondary cellular functionsimpaired by such pathologies.

Examples of diseases for which such treatment using the compound offormula (I) is useful include, but are not limited to, neuropsychiatricdiseases such as schizophrenia and related idiopathic psychoses,anxiety, sleep disorders, appetite disorders, affective disorders suchas major depression, bipolar disorder, and depression with psychoticfeatures, and Tourette's Syndrome, drug-induced psychoses, psychosessecondary to neurodegenerative disorders such as Alzheimer's orHuntington's Disease. It is anticipated that the compound of formula(I), a particularly selective inverse agonist of 5-HT2A that showslittle or no activity on dopaminergic receptors, may be especiallyuseful for treating schizophrenia. Treatment using the compound offormula (I) may also be useful in treating migraine, vasospasm,hypertension, various thrombotic conditions including myocardialinfarction, thrombotic or ischemic stroke, idiopathic and thromboticthrombocytopenic purpura, and peripheral vascular disease.

In a further embodiment the present invention provides methods fortreating or alleviating a disease condition associated with improperfunction, dysfunction, or stimulation of native, as well as titillatedor otherwise modified, forms of central or peripheral monoaminereceptors, such methods comprising administration of an effective amountof a compound of formula (I) to a host in need of such treatment. In oneembodiment, the monamine receptor is serotonin receptor in theperipheral nervous system, blood or platelets. In some embodiments, theserotonin receptor is a 5-HT2A subclass receptor. In additionalembodiments, the disease condition is associated with increased activityor activation of a serotonin receptor. Also provided are kits forperforming the same.

Some embodiments also pertain to the field of predictive medicine inwhich pharmacogenomics is used for prognostic (predictive) purposes.Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum, Clin Exp Pharmacol.Physiol., 23:983-985 (1996), and Linder, Clin. Chem. 43:254-66 (1997).In general, two types of pharmacogenetic conditions can bedifferentiated: genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action), andgenetic conditions transmitted as single factors altering the way thebody acts on drugs (altered drug metabolism). These pharmacogeneticconditions can occur as naturally occurring polymorphisms.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association,” relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map that consistsof 60,000-100,000 polymorphic or variable sites on the human genome,each of which has two variants). Such a high-resolution genetic map canbe compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high-resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1,000 bases of DNA.A SNP may be involved in a disease process; however, the vast majoritymay not be disease-associated. Given a genetic map based on theoccurrence of such SNPs, individuals can be grouped into geneticcategories depending on a particular pattern of SNPs in their individualgenome. In such a manner, treatment regimens can be tailored to groupsof genetically similar individuals, taking into account traits that maybe common among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a proteinor a receptor of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a molecule ormodulator of the present invention) can give an indication whether genepathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a molecule ormodulator of the invention, such as a modulator identified by one of theexemplary screening assays described herein. As we have describedpreviously, this approach can also be used to identify novel candidatereceptor or other genes suitable for further pharmacologicalcharacterization in vitro and in vivo.

Accordingly, one embodiment provides methods and kits for identifying agenetic polymorphism predisposing a subject to being responsive to thecompound of formula (I). The method comprises administering to a subjectan effective amount of the compound; identifying a responsive subjecthaving an ameliorated disease condition associated with a monaminereceptor; and identifying a genetic polymorphism in the responsivesubject, wherein the genetic polymorphism predisposes a subject to beingresponsive to the compound. It is anticipated that this method may beuseful both for predicting which individuals are responsive totherapeutic effects of the compound and also for predicting those likelyto experience adverse side effect responses. This approach may be usefulfor identifying, for example, polymorphisms in a serotonin receptor thatlead to constitutive activation and are thus amenable to inverse agonisttherapy. In addition, this method may be useful for identifyingpolymorphisms that lead to altered drug metabolism whereby toxicbyproducts are generated in the body. Such a mechanism has beenimplicated in the rare, but potentially life threatening side effects ofthe atypical antipsychotic, clozapine.

In a related embodiment, a method for identifying a subject suitable fortreatment with the compound of formula (I) is provided. According to themethod, the presence of a polymorphism that predisposes the subject tobeing responsive to the compound is detected, the presence of thepolymorphism indicating that the subject is suitable for treatment. Alsoprovided are kits for performing the same.

The compound of formula (I) preferably shows selective inverse agonistactivity towards the 5-HT2A receptor. Such activity is defined by anability of the ligand to attenuate or abolish the constitutive signalingactivity of this receptor. Selectivity in the present context isunderstood as a property of a compound of the invention whereby anamount of compound that effectively inversely agonizes the 5-HT2Areceptor and thereby decreases its activity causes little or no inverseagonistic or antagonistic activity at other, related or unrelated,receptors. In particular, the compound of formula (I) has surprisinglybeen found not to interact strongly with other serotonin receptors (5-HT1A, 1B, 1D, 1E, 1F, 2B, 2C, 4A, 6, and 7) at concentrations where thesignaling of the 5-HT2A receptor is strongly or completely inhibited. Inone embodiment, the compound is also selective with respect to othermonoamine-binding receptors, such as the dopaminergic, histaminergic,adrenergic and muscarinic receptors.

One embodiment of the present invention relates to a method ofalleviating or treating a disease condition in which modification ofmonoamine receptor activity, in particular 5-HT2A serotonergic receptoractivity, has a beneficial effect by administering a therapeuticallyeffective amount of the compound of formula (I) to a subject in need ofsuch treatment. Such diseases or conditions may, for instance arise frominappropriate stimulation or activation of serotonergic receptors. It isanticipated that by using a compound that is selective for a particularserotonin receptor subtype, in particular 5-HT2A, the problems withadverse side effects observed with the known antipsychotic drugs, suchas extrapyramidal effects, may be avoided substantially.

The term “therapeutically effective amount” as used herein means anamount of an active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation, amelioration, or lesseningof the symptoms of the disease being treated, or prevents or slows theprogress of the disease or increase of the symptoms.

In one embodiment, the compound of formula (I) may be administered in asingle daily dose, or the total daily dosage may be administered individed doses, for example, two, three or four times daily. Furthermore,the compound of formula (I) may be administered in intranasal form viatopical use of suitable intranasal vehicles, via transdermal routes,using those forms of transdermal skin patches well known to personsskilled in the art, by implantable pumps; or by any other suitable meansof administration. To be administered in the form of a transdermaldelivery system, for example, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

The dosage regimen utilizing the compound of formula (I) is selected inaccordance with a variety of factors including type, species, age,weight, sex and medical condition of the patient; the severity of thecondition to be treated; the route of administration; the renal andhepatic function of the patient; and the particular compound employed. Aphysician or veterinarian of ordinary skill can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the disease or disorder that is being treated.

For oral administration, compositions containing the compound of formula(I) are preferably provided in the form of tablets containing 0.01,0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0 or 50.0 mg of the activeingredient for the symptomatic adjustment of the dosage to the patientto be treated. In one embodiment, a unit dose contains from about 0.001mg to about 50 mg of the active ingredient. In another embodiment a unitdose contains from about 1 mg to about 10 mg of active ingredient.

The compound of formula (I) may be used alone at appropriate dosagesdefined by routine testing in order to obtain optimal pharmacologicaleffect on a monoaminergic receptor, in particular the 5-HT2Aserotonergic receptor subtype, while minimizing any potential toxic orotherwise unwanted effects. In addition, co-administration or sequentialadministration of other agents that improve the effect of the compoundmay, in some cases, be desirable.

In one embodiment, the compound of formula (I) may be combined with anadditional therapeutic agent. Additional therapeutic agents may include:levodopa (SINEMET™, SINEMET-CR™, bromocriptine (PARLODEL™), pergolide(PERMAX™), ephenedrine sulfate (EPHEDRINE™), pemoline CYLERT™), mazindol(SANOREX™), d,l-α-methylphenethylamine (ADDERALL™), methylphenydate(RITALIN™), pramipexole (MIRAPEX™), modafinil (PROVIGIL™), ropinirole(REQUIP™), an anti-dyskensia agent, an anti-dystonia, an anti-myoclonus,an anti-tremor agent, or an anti-psychotic agent. In some embodiments,the anti-dyskensia agent is selected from baclofen (Lioresal™),botulinum toxin (Botox™), clonazepam (Klonopin™), or diazepam (Valium™).In some embodiments, the anti-dystonia, anti-myoclonus, or anti-tremoragents are selected from baclofen (LIORESAL™), botulinum toxin (BOTOX™),clonazepam (KLONOPIN™), or diazepam (VALIUM™). In some embodiments, theanti-psychotic agent is selected from chlorpromazine (THORAZINE™),haloperodol (HALDOL™), molindone (MOBAN™), thioridazine (MELLARIL™), aphenothiazine, a butyrophenome, diphenulbutylpiperinde (pimozide),thioxanthines (fluphenthixol), substituted benzamides (sulpiride),sertindole, amisulpride, risperidone, clozapine, olanzapine,ziprasidone, aripiprazole, or their active metabolites(N-desmethylclozapine, N-desmethylolanzapine, 9-OH-risperdone)).

The pharmacological properties and the selectivity of the compound offormula (I) for specific serotonergic receptor subtypes may bedemonstrated by a number of different assay methods using recombinantreceptor subtypes, preferably of the human receptors if these areavailable, e.g. conventional second messenger or binding assays. Aparticularly convenient functional assay system is the receptorselection and amplification assay disclosed in U.S. Pat. No. 5,707,798,which describes a method of screening for bioactive compounds byutilizing the ability of cells transfected with receptor DNA, e.g.,coding for the different serotonergic subtypes, to amplify in thepresence of a ligand of the receptor. Cell amplification is detected asincreased levels of a marker also expressed by the cells.

Treatment of Neuropsychiatric Disorders

In one embodiment, the compound of formula (I) and related serotonin 2Aand/or 2C receptor inverse agonists alone or in combination with otherantipsychotic drugs, particularly those with dopamine antagonistproperties, are used to treat a variety of human neuropsychiatricdiseases including schizophrenia, schizoaffective disorders, mania andpsychotic depression. Specifically, the compound of formula (I) andrelated serotonin 2λ/2C receptor inverse agonists can improve psychoticsymptoms (feelings of being controlled by outside forces, hearing,seeing, smelling or feeling things which are not there, hallucinationsand unusual beliefs, delusions), negative symptoms (loss of normalbehavior including tiredness, loss of concentration and lack of energyand motivation, and cognitive function in psychotic patients when usedalone or in combination with other antipsychotic drugs. These agentsalso reduce the side-effects associated with the use of existingantipsychotic drugs and reduce the dose of existing agent that isrequired to achieve antipsychotic efficacy. Specifically, the compoundof formula (I) and related compounds alone or in combination withexisting antipsychotic drugs can be used to control the behavioral andneuropsychiatric manifestations present in all of these disease states.In some embodiments, pharmaceutical compositions comprised of acombination of the compound of formula (I) and existing antipsychoticagents are used.

Neuropsychiatric disorders associated with psychosis affect a largeproportion of the human population. Psychosis appears as a dominatingsymptom in diverse disorders, including schizophrenia, schizoaffectivestates, mania, psychotic depression among others. Current treatmentoptions primarily involve pharmacotherapy with a class of drugs known asantipsychotics. Antipsychotics are effective in ameliorating positivesymptomotology of these disorders, yet they frequently do not improveand may worsen negative and cognitive symptoms. Significant treatmentlimiting side effects are common with the use of antipsychotic drugs.

Drugs that possess antipsychotic properties have been in clinical usesince the early 1950's. Antipsychotic drugs are widely prescribed totreat psychotic symptoms irrespective of their etiology. Clinical use ofthese compounds is limited, however, by their side effect profiles.Nearly all of the “typical” or first generation compounds havesignificant adverse effects on human motor function. These“extrapyramidal” side effects, so termed due to their effects on humanmotor systems, can be both acute and chronic in nature. Acute effectsinclude dystonic reactions, and a potentially life threatening but raresymptom constellation; neuroleptic malignant syndrome. Chronic sideeffects include akathisias, tremors, and tardive dyskinesia. Due inlarge part to these disabling side effects, antipsychotic drugdevelopment has been focused on newer “atypical” agents (clozapine,olanzapine, quetiapine, risperidal, arapiprazole) that appear to havereduced liability for inducing adverse motoric effects. These newer“atypical” antipsychotic drugs, however, suffer from other limitingside-effects, including induction of cardiovascular abnormalities,extreme sedation, morbid obesity, type II diabetes, blood dyscrasias andpancreatitis among others.

While the precise molecular mechanisms mediating antipsychotic drugaction remain to be elucidated, antipsychotic drugs have been shown, byboth in vitro and in vivo methods, to interact with a large number ofcentral monoaminergic neurotransmitter receptors, includingdopaminergic, serotonergic, adrenergic, muscarinic, and histaminergicreceptors. It is likely that the therapeutic and adverse effects ofthese drugs are separable and are mediated by distinct receptorsubtypes.

Currently, it is thought that antipsychotic drugs reduce the positivesymptoms in these disorders by blocking dopamine D2 receptors. This isbased on the observation that these all antipsychotic drugs havereasonable affinity for this receptor in vitro, and that a correlationexists between their potency to block D2 receptors and their ability toreduce the positive symptoms of these disorders. Unfortunately, it islikely that antagonism of dopamine D2 receptors also mediates thedisabling extrapyramidal side effects.

The only other consistent receptor interaction that these drugs as aclass display is inverse agonism of 5-HT2A receptors, suggesting thatinverse agonism of these receptors is an alternate molecular mechanismthat confers antipsychotic efficacy. This theory is bolstered by anumber of basic scientific and clinical observations regardingserotonergic systems and the 5-HT2A receptor in particular (U.S. Pat.No. 6,358,698 incorporated by reference).

However, nearly all known antipsychotic agents lack specificity in theirmechanisms of action. In addition to possessing activity at dopamine D2receptors and 5-HT2A receptors, these drugs as a class have a multitudeof pharmacologically relevant interactions with critical neuronalproteins including a host of cell surface receptors, ion channels, andre-uptake transporters. This lack of drug target specificity likelycontributes to the multiplicity of adverse effects associated with useof existing antipsychotic agents.

These observations highlight the need to develop novel therapeuticregimens that are specifically designed to not only demonstrate efficacyagainst these particular disabling symptoms but to also possesstolerability in these specific patient populations. This can be achievedby improving the selectivity of the drug target interactions of newtherapeutic agents. Specifically, the development of agents with novelmechanisms of action that avoid the known pitfalls associated withexisting agents is desired. In addition, improved selectivity avoids theknown adverse effects associated with interactions with non-efficacyoff-target receptor interaction. For example many antipsychotic drugspossess high affinity interactions with H1 receptors. H1 antagonism isassociated with sedation. Further, other antipsychotic drugs haveaffinity interactions with alpha receptors. Antagonism of alpha-1receptors is associated with orthostasis. Improvements in therapeuticefficacy and safety also can be achieved by combining two or more agentseach with selective target interactions to achieve additive orsynergistic benefits. Specifically, by combining one drug thatspecifically interacts with D2 receptors as an antagonist and anotherdrug like the compound of formula (I) that interacts with specificallywith 5-HT2λ/2C receptors as antagonist or inverse agonist, the multitudeof off-target interactions of existing antipsychotic drugs can beavoided.

In one embodiment, serotonin 2A and/or 2C receptor inverse agonists areused to treat a variety of human neuropsychiatric diseases includingschizophrenia, schizoaffective disorders, mania, behavioral disturbancesassociated with dementia and psychotic depression. For example, thecompounds disclosed herein have utility in reducing the positivesymptoms, improving negative symptoms and enhancing cognitive functionin patients with certain neuropsychiatric diseases.

Antipsychotics and dopamine receptor antagonists can be effective inameliorating positive symptoms in schizophrenia and related diseases.Unfortunately, many of these compounds significantly worsen motorfunction and increase negative symptoms or leave these and othersymptoms untreated in these patients. Biochemical and pharmacologicaldata support the hypothesis that potentiation of serotonergicneurotransmission may be pathophysiologically important in thedevelopment of these unwanted effects and conversely blockade ofserotonergic neurotransmission may reduced the side-effects associatedwith antipsychotic drug therapy. While not being bound by this theory,the compound of formula (I) was selected to exploit the relationship ofserotonergic activity and the limiting effects associated withantipsychotic therapy.

Haloperidol is a typical antipsychotic with specificity as a D2 receptorantagonist. This compound commonly is used to treat the positivesymptoms associated with acute exacerbations of schizophrenia.Unfortunately, the use of this compound is associated with a plethora ofunwanted motoric side effects, including akathisia, parkinsonism,tardive dyskinesia and neuroleptic malignant syndrome. This compoundalso does not alter or worsens negative symptoms and cognitive functionin these patients.

In one embodiment, the compound of formula (I) can be used to treat manyside-effects that arise from antipsychotic therapy. For example, thecompound of formula (I) may be useful for treatment of motoricside-effects of other antipsychotic agents such as haloperidol. In oneembodiment, the compound of formula (I) is used for the treatment ofmotoric side-effects associated with haloperidol treatment.

In one embodiment, the compound of formula (I) may be usedprophylactically when for example, it is considered necessary toinitiate haloperidol therapy and it is feared that motoric deficits maydevelop.

In some embodiments, the compound of formula (I) may be used to treatpsychosis as a monotherapy or as an adjunct to medicaments to prevent ortreat antipsychotic drug side-effects caused by the medicament.Alternatively, the compound of formula (I) may be given in combinationwith other compounds, which also reduce antipsychotic drug side-effects.

In one embodiment, the compound of formula (I) may used to treat thenegative symptoms of certain neuropsychiatric disease includingschizophrenia as a monotherapy or as an adjunct to medicaments used totreat the positive symptom of these diseases.

In some embodiments, the compound of formula (I) also may used toimprove cognitive function in certain neuropsychiatric disease includingschizophrenia as a monotherapy or as an adjunct to medicaments used totreat the positive symptom of these diseases.

Methods of Preparation

The compound of formula (I) may be synthesized by methods describedbelow, or by modification of these methods. Ways of modifying themethodology include, among others, modification in temperature, solvent,reagents, etc.

The first step of the synthesis, illustrated below, is conducted in thepresence of acetic acid, NaBH₃CN, and methanol to produce the compoundof formula (II):

The compound of formula (IV) can be synthesized by treatment of thecompound of formula (III) with isobutyl bromide and potassium carbonatein dimethyl formamide (DMF) at about 80° C.:

The compound of formula (IV) can be converted to the compound of formula(V) by reaction with potassium hydroide in methanol/water:

The compound of formula (V) is heated to reflux with diphenylphosphonylazide (DPPA) and a proton sponge in tetrahydrofuran (THF) to produce thecompound of formula (VI):

Finally, reaction of the compound of formula (II) with the compound offormula (VI) in methylene chloride produces the compound of formula (I):

The tartrate salt of the compound of formula (I) may be produced bymixing with L-(+)-Tartaric acid in ethanol:

EXAMPLES

The examples below are non-limiting and are set forth to illustrate someof the embodiments disclosed herein.

Example 1 Agonist Studies

Parkinson's disease is typically managed using direct acting dopamineagonists. Examples of this class of compounds include pergolide,bromocriptine, pramipexole and ropinirole. These drugs are thought to beeffective because of their agonist activity at the dopamine D₂, D₃, andD₄ receptors located in striatal and forebrain regions. This activitymay compensate for the progressive loss of forebrain dopaminergicinnervation that characterizes the PD. However, these drugs are notspecific for these dopaminergic receptors and also possess potentagonist activity at other receptors, including 5HT2A and 5HT2Creceptors. Using a physiologically predictive in vitro functional assay,it is shown below that pergolide, lisuride, and bromocriptine displayagonist potencies at human 5HT2A receptors that are equivalent to thoseobserved at the human D₂ receptor. (FIGS. 1A and 1B, and Table 1).

Using the R-SAT assay, the activity of common dopeaminergic compoundsagainst dopamine and serotonin receptor types was studied. (See U.S.Pat. Nos. 5,912,132 and 5,955,281, both of which are hereby incorporatedby reference.) In FIG. 1, data were plotted as percentage agonistresponse as determined for a reference full agonist (100%) versus drugconcentration. The reference full agonist used for the D₂ receptor wasquinpirole, while serotonin was used for the 5HT2A receptor. Compoundstested include dopamine (filled squares), quinpirole (filled circles),lisuride (filled triangles), bromocriptine (filled diamonds), serotonin(open squares), and pergolide (filled inverted triangles). Potencies ofrepresentative dose response curves using dopamine D₂ receptors weredetermined and are shown in FIG. 1A; (pergolide-0.2 nM, dopamine-8.0 nM,lisuride-0.023 nM, quinpirole-3.3 nM, bromocriptine-0.43 nM, andserotonin-no response).

FIG. 1B shows compound potency against the serotonin 5HT2A receptor;(dopamine-no response, quinpirole-174 nM, lisuride-0.028 nM,bromocriptine-2.7 nM, serotonin-33 nM, and pergolide-0.22 nM).

Because these drugs are administered in the clinic to achieve D₂receptor occupancy, these data argue that direct acting dopamineagonists are also behaving as 5HT2A receptor agonists in vivo whenadministered in therapeutic doses to PD patients.

TABLE 1 Serotonin Receptor Agonist Activity of Dopaminergic Agents Usedin PD Drug Dopamine D2 Serotonin 2A Serotonin 2C Dopamine 8.40 +/− 0.32NA NA Serotonin NA 7.73 +/− 0.04 7.29 +/− 0.10 Lisuride 11.00 +/− 0.36 10.65 +/− 0.10  7.61 +/− 0.13 Pergolide 9.45 +/− 0.06 8.05 +/− 0.22 6.66+/− 0.08 Bromocriptine 9.30 +/− 0.31 8.75 +/− 0.14 5.80 +/− 0.05Ropinirole 8.19 +/− 0.58 6.85 +/− 0.77 NT Pramipexole 8.15 +/− 0.38 5.93+/− 0.74 NT Apomorphine 6.24 +/− 0.11 NA NA

Data are derived from R-SAT assays. As shown, all compounds displayedfull (>75%) relative agonist efficacies. Data are reported as −Log(EC₅₀)values+/−standard deviation of three to eight separate determinations.The VGV isoform of the 5HT2C receptor, and the short form of the D₂receptor were utilized for these studies. NA denotes no activity, NTdenotes not tested.

The agonist activity of these anti-parkinsonian agents at human 5HT2A/Creceptors has particular implications for the generation and treatmentof human hallucinations and psychosis. That certain natural andsynthetic chemical compounds can induce hallucinatory states in humanshas led to detailed investigations of the mechanisms of action of thesehallucinogenic or psychotomimetic drugs. These efforts have implicated anumber of molecular activities of these classes of drugs as beingrelevant to their ability to induce hallucinations, particularly visualhallucinations, in normal healthy individuals. Hallucinogens fall intotwo distinct chemical classes, the phenylethanolamines, and thesubstituted tryptamines, both of which are structurally related toserotonin. Many in vitro studies, utilizing radioligand bindingtechniques, as well as functional pharmacological assays, haverepeatedly demonstrated that these drugs are potent 5HT2A and 5HT2Creceptor agonists (5). More recent in vivo studies, in which normalvolunteers are administered the hallucinogen MDMA (Ecstasy) and thenevaluated for clinical response, as well as anatomical measures of brainactivation utilizing functional neuro-imaging technologies, havedemonstrated that the psychometric and pharmacological activities ofhallucinogens can be blocked by anti-psychotic drugs as well as thecompound ketanserin (6,7). These drugs share a common molecularproperty, 5HT2A receptor inverse agonism.

Example 2 Inverse Agonist Studies

Once treatment-induced motoric and neuropsychiatric symptoms develop inPD patients, few viable therapeutic options exist to manage thesedisturbances. Treatment strategies differ for these two classes ofsymptoms, but one uniformly clinically efficacious, yet poorly toleratedapproach, involves the use of antipsychotic agents. Antipsychotics areknown to possess high affinity for the dopamine D₂ subclass of dopaminereceptors and neutral antagonism of these receptors underlie thetherapeutic efficacy of these drugs in human psychosis. In addition todopamine D₂ receptor antagonism, these agents Possess a wide range ofadditional potent and pharmacologically relevant activities at many ofthe other monoaminergic receptor subtypes including serotonin,adrenergic, muscarinic and histaminergic receptors. Of these additionalmolecular actions, 5HT2A receptor interactions have been the subject ofsignificant study. That antipsychotics have high affinity for multiplereceptor subtypes, including serotonin 2 receptors, was demonstrated bythe application of radioligand binding techniques (8). The methodologiesused to document this cannot define the nature of the interactionbetween an anti-psychotic antipsychotic and a given receptor. Forexample, the methods are unable to distinguish as to whether a drugpossesses positive (agonist) or negative (inverse agonist) intrinsicactivity, or if it lacks intrinsic activity and functions as a neutralantagonist. Recently, this class of drugs was profiled using afunctional assay that can discriminate the mechanistic nature of adrug-target interaction (9).

This approach revealed a number of novel aspects of antipsychotic drugaction (See U.S. Pat. No. 6,358,698, which is hereby incorporated byreference in its entirety). It confirmed that these drugs as a classpossess potent neutral antagonistic activity at the D₂ receptor.Importantly, it also revealed that nearly all antipsychotic drugs, withthe exception of the substituted benzamides, possess potent negativeintrinsic activity (inverse agonism) at the 5HT2A receptor. Theseefforts have identified inverse agonist activity at the 51-IT2A receptoras being a critical molecular component of anti-psychotic drug action,and suggest that compounds that are selective 5HT2A receptor inverseagonists may have antipsychotic efficacy, even in the absence of D₂receptor activity.

None of the older typical antipsychotics, exemplified by haloperidol,can be administered to PD patients because of severe worsening in theirmotor states. The more recent development of newer atypical agents,namely those with reduced (but clearly not absent) liability to inducedmotoric side effects, suggested that perhaps these agents could be usedin PD patients to control dyskinesias and hallucinosis. Unfortunately,the majority of these agents are not tolerated in PD patients secondaryto worsening of motor function (10). Of the atypical agents, only one,clozapine, has shown efficacy in treating these adversetreatment-induced side effects in PD patients without untoward motoricliabilities. As such, an improved understanding of the in vitromolecular profile of clozapine can provide critical insights into thedesign of novel agents for these difficult to treat indications.

The demonstration that clozapine is tolerated in PD patients comes fromstudies on treatment-induced psychosis. Two well-designed placebocontrolled, double blind clinical trials have shown that clozapine isefficacious in psychotic PD patients, and does not worsen parkinsonism,at doses in the 25-35 mg/day range (11,12). Similarly, two open labelstudies of clozapine in L-dopa and apomorphine induced dyskinesias alsodemonstrate efficacy and tolerability of low doses of clozapine, on theorder of 50-100 mgs/day in these patients (13,14). The dosages used inthese PD patients are much lower than the typical 600-900 mg/day rangeof doses used in treatment refractory schizophrenia. Commensurate withthis lower dosing, plasma levels of clozapine in PD patients withpsychosis ranged from 4.5 to 16.1 ng/ml (15). This is dramatically lowerthan the 250 ng/ml average serum levels that are associated withtherapeutic response in refractory schizophrenic patients.

Not surprisingly, the administration of low dose clozapine, and thecommensurate plasma levels obtained at these doses, are well below thosenecessary for D₂ receptor occupancy, providing a mechanisticunderstanding of why these dosages are tolerated with respect to motoricliability in these patients. (Positron emission tomography (PET) studiesin schizophrenic patients have defined steady state plasmaconcentrations of clozapine that are required to generate high occupancyof striatal dopamine D₂ receptors). These data also argue that efficacyin dyskinesia and psychosis is mediated by one or more of the non-D₂receptor targets of this drug. Since rank orders of receptor potencies,as determined by in vitro pharmacological assays, has repeatedly beenshown to be a reliable predictor of in vivo receptor action, thereceptor sites for which clozapine display a higher potency than D₂receptors would be predicted to potentially mediate its clinicalefficacy in this indication. Detailed functional profiling of clozapineagainst over 30 of the known monoaminergic receptor subtypes hasidentified only five sites with higher affinity than dopamine D₂receptors, histamine H1, muscarinic m1 and m4, and serotonin 2A, 2B, and6 receptors. Table 2 reports the absolute and relative potencies ofclozapine at some of these monoamine receptor targets as determined bythe physiologically predictive in vitro R-SAT assay. These data suggestthat at the clinical dosing and serum levels of clozapine observed inPD, two receptor sites are preferentially occupied, the histamine H₁ and5HT2A receptors.

Conversely, plasma levels achieved with 50 mgs/day of clozapine resultin full occupancy of cortical 5HT2A receptors, and extrapolation to theplasma levels observed in PD patients treated for psychosis suggest nearcomplete occupancy of 5HT2A receptors at these dosages as well (16).Whereas central occupancy of 5HT2A receptors, coupled with negativeintrinsic activity, may mediate efficacy in these states, centraloccupancy of histamine H₁ receptors is known to cause sedation, aneffect that was observed in the majority of PD patients treated with lowdose clozapine. Taken together these data suggest that clozapine isacting primarily as a 5HT2A receptor inverse agonist in this clinicalsetting.

TABLE 2 Antagonist and Inverse Agonist Potencies of Clozapine atMonoamine Receptors D₂ 5HT2A 5HT2B 5HT2C H₁ Clozapine 72 +/− 56 6.4 +/−1.0 20 +/− 9 250 +/− 60 0.40 +/− 0.07 Ratio to D₂ 11 3.6 0.3 180

Data are derived from (9) and are reported as Ki values for the D2receptor determined as a competitive antagonist, and EC₅₀ values for theremaining receptors determined as inverse agonists, in nanomolarunit's+/−standard deviation of three to eight ‘separate determinations.

Behavioral Pharmacological Evidence

The tartrate salt of the compound,N-(1-methylpiperidin-4-yl)-N-(4-fluorophenylmethyl)-N’-(4-(2-methylpropyloxy)phenylmethyl)carbamide(compound of formula (I)), is a potent, selective, orally bioavailable5HT2A receptor inverse agonist. The compound of formula (I) alsopossesses lesser potency as a 5-HT2C receptor inverse agonist and lacksintrinsic activity at the remaining monoaminergic receptor subtypes.Perhaps most notably, the compound of formula (I) lacks activity atdopamine receptor subtypes. (See U.S. patent application Ser. No.09/800,096, which is hereby incorporated by reference in its entirety).Extensive behavioral pharmacological profiling of this agent, includingpre-clinical models of antipsychotic and anti-dyskinetic drug actionssupport the therapeutic use of the compound of formula (I) inParkinson's Disease and related human neurodegenerative diseases.

Example 3 Animal studies

To determine potential in vivo antipsychotic activity, we studied thecompound of formula (I) in an animal model that predicts such efficacyin humans. The compound of formula (I) attenuates hyperactivity inducedby the non-competitive N-methyl-d-aspartate (NMDA) antagonist MK-801(dizocilpine) with a minimum effective dose of 1 mg/kg s.c. (FIG. 2A),and 10 mg/kg p.o. (FIG. 2B). The compound of formula (I) also reducedspontaneous locomotion at 3 mg/kg and higher s.c. doses (FIG. 2A), andat oral doses between 10 and 100 mg/kg (FIG. 2B). In FIGS. 2A and 2B,asterisks indicate statistical significance (p<0.05) compared torespective vehicle control. Inhibition of MK-801 is a property shared bymost atypical antipsychotic agents, and after i.p. administration, thecompound of formula (I) attenuated MK-801 hyperactivity at 1 mg/kg, in amanner similar to the atypical antipsychotic clompine.

Example 4 Primate animal studies

To determine the potential in vivo anti-dyskinetic activity, we studiedthe compound of formula (I) in an animal model that predicts suchefficacy in humans. The use of1-methyl-4-phenyl-1,2,3,6-tetrahydropyrilidine (MPTP) to induceparkinsonism in monkeys, coupled with prolonged administration of L-dopainduces severe dyskinesias. The compound of formula (I), whenadministered s.c., to dyskinetic primates was found to significantlydiminish L-dopa induced dyskinesias in a dose dependent manner asdetermined by the reduction of observable dyskinetic movements scored asa percentage of those present in placebo injected animals (FIG. 3).

Example 5 5HT2A/C Serotonin Antagonist Treatment of Parkinson's Disease

The present example demonstrates that blockage of 5HT2A/C receptors withthe compound of formula (I) in parkinsonian patients reduceslevodopa-associated dyskinesias and motor response fluctuations.Additionally, the compound of formula (I) is shown to be safe andtolerated at effective doses and potentiates the beneficial effects oflevodopa on parkinsonian symptoms.

The compound of formula (I) is administered orally in a group of 21parkinsonian patients in a double blind, placebo controlled studylasting approximately 5 weeks. An unbalanced parallel-group doseescalation design is used involving an initial placebo run-in, followedby a randomized (active) phase of the compound of formula (I) orplacebo. The compound of formula (I) is administered once daily for fourweeks, with the dose escalating once each week. Assessments are made onthe first day of each dose escalation.

The study is conducted on an outpatient basis. Studies of the compoundof formula (I) effect on the motor response to levodopa are conducted inaccordance with the standard Experimental Therapeutics Branch (ETB)paradigm, which makes use of a steady state infusion of dopaminomimeticsin order to maximize the reliability of data acquisition as well as topermit determination of the anti-parkinsonian efficacy half-time.

Patients who participate in the study have particular characteristics.The patients are between 30 and 80 years of age, inclusively. Thepatients had been diagnosed with idiopathic Parkinson's disease based onthe presence of a characteristic clinical history and neurologicalfindings. The patients displayed relatively advanced disease symptomswith levodopa-associated motor response complications, includingpeak-dose dyskinesias and wearing-off fluctuations.

The sample size is calculated for the primary endpoint; the UnifiedParkinson's Disease Rating Scale (UPDRS) part III motor examination. Asample size of 17 provides 80% power to detect predicted differences, a40% reduction, with a standardized effect size of 1, using a two-tailedt-test at the 0.05 significance. This assumes an anti-dyskinetic effectof the compound of formula (I) to be compared to that of amantadine (asobserved in previous ETB studies), and a linear dose-response of thecompound of formula (I). In this phase 2 study we will accept atwo-sided alpha at a 0.05 significance level. Four patients will beadded for the placebo group, totaling 21 subjects enrolled in the study.

Patients enter the levodopa infusion optimal rate determination (dosefinding) portion of the study as soon as all prohibited medication hasbeen withdrawn for at least four weeks. If the patient has had anintravenous dosing rate for levodopa optimized within the past threemonths, these doses may be used for the study.

Intravenous infusion of levodopa is conducted in an in-patient ward. Onthe night prior to all infusions, subjects' usual anti-parkinsonianmedications are withheld (levodopa by 12 AM, dopamine agonists by 6 PM).During the first and second days of optimal rate determination, twobaseline UPDRS ratings are performed prior to levodopa infusion.Initially, the “optimal” rate of levodopa infusion is carefully titratedfor each individual to determine the minimum dose needed to achieve astable “on” state characterized by an “optimal” reduction inparkinsonian signs and mild but ratable dyskinesias (comparable topatient's usual “on” state). Dyskinesia severity is similar to thatexperienced with each patient's usual therapeutic regimen. Levodopa willbe administered by means of an indwelling intravenous catheter. Theinitial infusion rate of levodopa will not exceed 80 mg/hr. Subsequentinfusion rates may be gradually increased until the optimal rate isfound, up to a maximum of 2 mg/kg/hour.

Levodopa infusions will ordinarily last up to 8 hours, but may becontinued uninterrupted for several days or be repeated on other days toobtain reliable assessment of motor function. The peripheraldecarboxylase inhibitor carbidopa (50 mg, given every 3 hours) isadministered orally starting at least one hour prior to intravenousadministration of levodopa and continuing until levodopa effects haveworn off. After the initial “optimal” rate finding for levodopainfusion, all subsequent infusions are given at the predetermined“optimal rate”. As an intravenous levodopa formulation is notcommercially available in this country, is administered under ETB IND22,663.

Patients are dosed according to Table 3:

TABLE 3 Patient group Week 1 Week 2 Week 3 Week 4 Week 5 I PlaceboPlacebo Placebo Placebo Placebo II Placebo 30 mg 70 mg 150 mg 300 mgCompound Compound Compound Compound (I) (I) (I) (I)

Patients proceed through this dose escalation scheme until week 5 oruntil maximum tolerated dose is attained.

Throughout the study, patients are evaluated weekly for drug safety andtolerability during their inpatient admission and two weeks aftertreatment for an outpatient follow-up visit. During each inpatientadmission, patients remain under close medical monitoring by staffphysicians and nurses. If at any time during the treatment period, thestaff physician determines that a patient does not tolerate any givendose, the patient will be considered to have attained maximum tolerateddose and will not receive any additional doses of the compound offormula (I). Patients are encouraged to contact study staff betweenstudy days to report any adverse experiences.

Patients are observed in the hospital and will not be discharged untilfree of all significant adverse effects, if any. Safety assessments,which are performed on study days, include adverse experiences,monitoring vital signs, standard safety monitoring, and cardiacmonitoring.

Subjects in Patient Group II show a reducing in levodopa-associateddyskinesias and motor response fluctuations. The subjects in PatientGroup II tolerate the compound of formula (I) at all doses administered.The compound of formula (I) therapy also potentiates the beneficialeffects of levodopa on parkinsonian symptoms.

Example 6 R-SAT Assay

The functional receptor assay Receptor Selection and AmplificationTechnology (R-SAT) was used to investigate the activity of the compoundof formula (I) as an inverse agonist at 5HT2A receptors. The compound offormula (I) exhibited high potency (pIC50 of 9.1) and high efficacy(98%) at 5HT2A receptors.

Example 7 Anti-Psychotic Activity Study

To determine potential in vivo antipsychotic activity, we studied thecompound of formula (I) in an animal model that predicts such efficacyagainist positive symptoms in humans (FIG. 4). In FIG. 4, ACP refers tothe compound of formula (I). The compound of formula (I) did not reducehyperactivity induced by 3.0 mg/kg I.P. of the indirect dopamine agonistd-amphetamine when administered alone at doses of 10.0 mg/kg P.O. andbelow to mice. As expected, haloperidol dose-dependently reducedamphetamine hyperactivity with a minimally significant effect seen at0.1 mg/kg, s.c. When a 10.0 mg/kg P.O. dose of the compound of formula(I) was administered in combination with various s.c. doses ofhaloperidol, the minimally significant dose of haloperidol was decreasedto 0.03 mg/kg. With this combination, amphetamine hyperactivity iscompletely reversed. Thus, an inactive dose of the compound of formula(I), when combined with an inactive dose of haloperidol produces acomplete reversal of amphetamine hyperactivity. This suggests that theantipsychotic activity of haloperidol may be significantly enhanced whenit is combined with the compound of formula (I). Equally important, whenthe compound of formula (I) is combined with haloperdiol, the dose ofhaloperidol can be lowered without a loss of efficacy. This would beexpected to improve the safety margin for the clinical use ofhaloperidol in neuropsychiatric diseases.

LITERATURE CITED

The following references are incorporated herein by reference in theirentireties.

-   1. Everett, G., M., and Borcherding, J., W. (1970) L-dopa: effect on    concentration of dopamine, norepinephrine and serotonin in brains of    mice. Nature, 168: 849-850.-   2. Butcher, L., Engel, J., and Fuxe, K. (1970) L-dopa induced    changes in central monoamine neurons after peripheral decarboxylase    inhibition. J. Pharm. Pharmac., 22: 313-316.-   3. NG, K., Y., Chase, T., N., Colburn, R., W., and Kopin,    I., J. (1970) L-dopa induced release of cerebral monoamines.    Science, 170: 76-77.-   4. Birkmayer. W., Danielczyk, W., Neumayer, E., and    Riederer, P. (1974) Nucleus Ruber and L-dopa Pstchosis: Biochemical    Post mortem findings. Journal of Neural Transmission, 35; 93-116.-   5. Sadzot, B., Baraban, J., M., Glennon, R., A., Lyon, R., A.,    Leonhardt, S., Jan, C., R., and Tietler, M. (1989) Hallucinogenic    drug interactions at human brain 5-HT2 receptors; implications for    treating LSD-induced hallucinogenesis. Psychopharmacology, 98(4):    495-499.-   6. Liechti, M., E., Geyer, M., A., Hell, D., and Vollenwieder,    F., X. (2001) Effects of MDMA (ecstasy) on prepulse inhibition and    habituation of startle in humans after pretreatment with citalopram,    haloperidol, or ketanserin., Neuropsychopharmacology, 24(3):    240-252.-   7. Gamma, A., Buck, A., Berthold, T.; Liechti, M., E., and    Vollenweider, F., X. (2000) 3,4-methylenedioxymethamphetamine (MDMA)    modulates cortical and limbic brain activity as measured by    [H(2)(15)O]-PET in healthy humans., Neuropsychopharmacology, 23(4):    388-395-   8. Leysen, J., E., Niemegeers, C., J., Tollenaraere, J., P., and    Laduron, P., M. (1978) Serotonergic component of neuroleptic    receptors. Nature (Lond) 272: 168-171.-   9. Weiner, D., M., Burstein, E., S., Nash, N., Croston, G., E.,    Currier, E., A., Vanover, K., E., Harvey, S., C., Donohue, E.,    Hansen, H., C., Andersson, C., M., Spalding, T., A., Gibson, D., F.,    Krebs-Thomson, K., Powell, S., B., Geyer, M., A., Hacksell, U., and    Brann, M., R. (2001) 5-hydroxytryptamine 2A receptor inverse    agonists as antipsychotics. J Pharmacol Exp Ther. 299(1): 268-76.-   10. Friedman, J., H., and Factor, S., A. (2000) Atypical    antipsychotics in the treatment of drug-induced psychosis in    Parkinson's disease. Mov. Disord, 15(2): 201-211.-   11. The Parkinson Study Group (1999) Low-dose clozapine for the    treatment of drug-induced psychosis in Parkinson's disease. New    Eng. J. Med., 340(10): 757-763.-   12. The French Clozapine Study Group (1999) Clozapine in    drug-induced psychosis in Parkinson's disease. Lancet, 353:    2041-2042.-   13. Bennett, J., P., Landow, E., R., and Shuh, L., A. (1993)    Suppression of dyskinesias in advanced Parkinson's Disease. II    Increasing daily clozapine doses suppress dyskinesias and improve    parkinsonism symptoms. Neurology, 43: 1551-1555.-   14. Durif, F., Vidailhet, M., Assal, F., Roche, C., Bonnet, A., M.,    and Agid, Y. (1997) Low-dose clozapine improves dyskinesias in    Parkinson's disease. Neurology, 48: 658-662.-   15. Meltzer, H., Y., Kennedy, J., Dai, J., Parsa, M., and    Riley, D. (1995) Plasma clozapine levels and the treatment of    L-DOPA-induced psychosis in Parkinson's disease. A high potency    effect of clozapine. Neuropsychopharmacology, 12(1): 39-45.-   16. Nordstrom, A., L., Farde, L., and Halldin, C. (1993) High 5-HT2    receptor occupancy in clozapine treated patients as demonstrated by    PET. Psychopharmacology, 110(3): 365-367.-   Bibbiani, F., Oh, F., D., and Chase, T., C. (2001) Serotonin 5-HT1A    agonist improves motor complications in rodent and primate    parkinsonian models. Neurology, 57: 1829-1834.

1.-16. (canceled)
 17. A method of treating Lewy Body Dementia in apatient, comprising administering to a patient in need of such treatmenta therapeutically effective amount of a compound of formula (I):

or a pharmaceutically acceptable salt thereof.
 18. The method of claim17, wherein the salt is a tartrate salt.
 19. The method of claim 17,wherein the amount of the compound of formula (I), or a pharmaceuticallyacceptable salt thereof, is from about 0.001 mg to about 50 mg.
 20. Themethod of claim 17, wherein the amount of the compound of formula (I),or a pharmaceutically acceptable salt thereof, is from about 1 mg toabout 10 mg.
 21. The method of claim 17, wherein the amount of thecompound of formula (I), or a pharmaceutically acceptable salt thereof,is about 10 mg.
 22. The method of claim 17, wherein the amount of thecompound of formula (I), or a pharmaceutically acceptable salt thereof,is about 25 mg.
 23. The method of claim 17, wherein the amount of thecompound of formula (I), or a pharmaceutically acceptable salt thereof,is about 50 mg.
 24. The method of claim 17, wherein the compound offormula (I) is administered daily.
 25. The method of claim 17, whereinthe compound of formula (I) is administered once daily.
 26. The methodof claim 17, wherein the compound is administered in combination with ananti-psychotic agent.
 27. The method of claim 26, wherein theanti-psychotic agent selected from the group consisting ofchloropromazine, haloperidol, molindone, thioridazine, a phenothiazine,a butyrophenone, diphenylbutylpiperidine, a thioxanthine, fluphenthixol,a substituted benzamide, sulpiride, sertindole, amisulpride,risperidone, clozapine, olanzapine, ziprasidone, aripirazole,N-desmethylclozapine, N-desmethylolanzapine, and 9-OH-risperidone. 28.The method of claim 26, wherein the anti-psychotic agent is haloperidolor risperidone.